System for stabilizing samples

ABSTRACT

A transport/preservation system has been found which provides safe, effective transport for patient specimens and bodily fluids, and functions as a preservative for biological reagents, therapeutics, and personal care products. The transport/preservative formulations can also be used as safe, effective sanitizers on surfaces, equipment, and appliances. The transport preservation formulation includes a biguanide and one or more other antimicrobial agents, which is cidal to microorganisms when present in a sample or on a surface to be sanitized.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of application Ser. No.10/048,660, filed May 7, 2002, now U.S. Pat. No. 6,716,632 issued onApr. 6, 2004, the entire contents of which are hereby incorporated byreference, which was filed under 35 U.S.C. 371, based on InternationalApplication No. PCT/US00/10384, filed Apr. 18, 2000, and claiming thebenefit of U.S. Provisional Application No. 60/146,729, filed Aug. 2,1999.

TECHNICAL FIELD

This application relates to stabilization and preservation of patientspecimens, bodily fluids, therapeutics, personal care products, surfacesanitizers or biological reagents.

BACKGROUND OF THE INVENTION

Diagnostic laboratory tests on patient bodily fluids or tissue specimensprovide significant information for disease treatment. These tests aidin discovery of occult disease, early diagnosis after the onset of signsand symptoms, differential diagnosis of various possible diseases,determination of the stage of the disease, estimation of the activity ofthe disease, detection of recurrence of disease, and measurement of theefficacy of therapy. The diagnostic benefits of many laboratory tests,however, require that the chemical and physical properties of apatient's sample remain substantially unaltered during transport, i.e.,the time required to go from sample collection from the patient toprocessing and/or testing by the laboratory.

Microbial contamination of a patient's sample can cause significantchanges to its physical and biochemical composition during transport. Ifthe bioburden of a sample is low and the sample is processed within ashort time (2 to 4 hours) after collection, the microorganisms presentusually will not affect the sample's chemical properties. However, ifthe sample can sustain the growth of organisms and the time betweencollection and processing exceeds 4 hours, the growth and/or metabolismof the microorganisms can alter the chemical and/or physical propertiesof the sample. For example, the organisms may be able to consume certaincomponents present in the sample such as carbon, nitrogen, or minerals,thus, removing or altering components which were present in the sampleat the time of collection. Secondly, as a result of microbial growth,metabolism or death, components which were not present at the time ofcollection may be released into the sample.

One type of patient sample which is greatly affected by microbialcontamination during transport is a urine sample submitted forurinalysis. Urinalysis is a series of tests performed on the urinesample including leukocytes, cast cells, red blood cells, glucose,bilirubin, ketone, specific gravity, pH, protein, urobilinogen, nitrite,and blood. Normal healthy women have 10²-10³ microorganisms present intheir urine. Microorganisms can also be introduced into the patient'surine sample either through a patient's clinical infection or byinadvertent contamination during the collection process or transport tothe laboratory. Since urine samples generally contain sufficientmetabolites and other factors required to support the growth andreplication of most microorganisms commonly found in urine samples,delays in transport beyond 4 hours can lead to significant changes inthe chemical and physical properties of the samples. For example, in onestudy, common urinary tract contaminants or potential urinary pathogenssignificantly altered the chemical/physical properties of unpreservedurine held for 8-24 hours or longer at room temperature: false positivereactions for hemoglobin, protein, nitrites, and esterase; falsenegative reactions for nitrites, glucose, protein and ketones; andsubstantial changes in pH. Many of these alterations in the chemicalproperties of the unpreserved urine samples occurred within 8 hours(Dorn, G. L., unpublished).

Laboratory standards recommend that urine samples be analyzed within twohours after collection from the patient to circumvent changes inchemical and physical properties. However, with the emergence of HealthMaintenance Organizations (HMO's), Preferred Physician Organizations(PPO's), and centralized laboratory testing facilities combined withincreasing pressure to be more cost effective through batch processingof samples, it has become increasingly difficult to comply withtraditional standards of practice. For example, one study showed that alarge percentage of samples submitted for culture within a hospitalsetting were>4 hours old prior to processing. (Dorn, et al., “Adherenceto laboratory guidelines: a study on urine specimen transport time,”Diagnostics & Clinical Testing 27:28-31 (1989)). Another study indicatedthat all samples submitted to centralized commercial laboratoriesexceeded the recommended time limits for transport. (Dorn, G. L.,“Microbial stabilization of antibiotic-containing urine samples by usingthe FLORASTAT urine transport system,” J Clin Micro 29:2169-2174(1991)). In the area of urinalysis, a recent College of AmericanPathologist Q-Probes Study documented that for inpatients andoutpatients, respectively, only 64% and 77% of laboratories were able tomeet the 2-hour transport goal 90% of the time. (Howanitz, et al.,“Timeliness of urinalysis: a College of American Pathologists Q-probesstudy of 346 small hospitals,” Arch Pathol Lab Med 121:667-672 (1997)).

Since current medical practices often prevent expedient transport ofurine samples for urinalysis, and since microorganisms are frequentlypresent in urine samples collected for urinalysis, a method of blockingthe deleterious effects of microbial contamination on a urine sampleduring transport is advantageous in preserving the chemical and physicalintegrity of the sample. For this purpose, various preservatives forurine samples have been developed and commercialized. Among the activeingredients for these preservatives are: boric acid, mercuric oxide,sodium azide, tartaric acid, and thimerosal (ethylmercurithiosalicylicacid). Boric acid has been reported to be compatible as a preservativeused in combination with urinalysis and leukocyte/cast cell analysis.(Porter, I. A. and Brodie, J. 1969. “Boric acid preservation of urinesamples,” Br Med J 2:353-355; Guenther, K. L. and Washington II, J. A.1981. Evaluation of the B-D urine culture kit,” J Clin Microbiol14:628-630).

Despite the commercial availability of preservatives for urine samples,there are significant deficiencies associated with them. For example,many of the active ingredients used in commercially available urinepreservative systems present health, flammability, and/or reactivityhazards. According to the National Fire Prevention Association's health,flammability, and reactivity hazard ratings for chemicals, a shortexposure to mercuric oxide and thimerosal could cause death or majorresidual injury. Although mercury-based systems such as mercuric oxide(Starplex Scientific, Inc., Etobicoke, Ontario, Canada) and thimerosal(Sigma Chemical Co., St. Louis, Mo.) effectively stabilize samples,their high National Fire Protection Association (NFPA) health hazardrating makes them unsuitable for use when large numbers of samples orhigh volumes of material are being processed. While sodium azide (SigmaChemical Co.) is also an effective stabilizing material, the healthrating assigned to sodium azide indicates that short exposure couldcause serious temporary or residual health injury, making it unsuitablefor use in high volume processing. While boric acid (Becton Dickinson,Franklin Lakes, N.J.) (Sage, Inc., Crystal Lakes, Ill.) (Bibby Sterlin,Ltd., Dynalab Corp., Rochester, N.Y.) and tartaric acid (Mid-AmericaHealth, Niagara Falls, N.Y.) have moderate to low NFPA hazard ratings,they are not cidal and, consequently, do not effectively block thedeleterious effects of all microorganisms of interest, potentiallycausing false negative and false positive urinalysis results when theurine sample is held at room temperature beyond 8 hours. Therefore, asillustrated in the case of urine samples submitted for urinalysis, thereis a continuing need for an effective transport system which providesstability of the chemical and physical properties of patient specimensand bodily fluids without exposing patients, healthcare professionals,and laboratory personnel to serious health hazards.

Biological reagents, some of which are often used in diagnostic testingprocedures, are also susceptible to chemical and physical alteration dueto microbial contamination. These reagents contain substances which arecritical to their function but also capable of supporting microbialgrowth and/or metabolism. Although most biological reagents aremanufactured under sterile conditions in sealed containers, low levelmicrobial contamination can occur during manufacturing. During storage,the growth of the contaminating microorganisms can cause chemical andphysical changes to the reagent. Moreover, many reagents are sold inmultiple-entry containers at volumes which allow the user torepetitively extract small aliquots over time. There exists thepossibility of microbial contamination of the reagent at each entryevent. One commercially available preservative for reagents isMicr-O-protect™ (Roche Diagnostics, GmbH, Mannheim, Germany), anethanolic solution of bromonitrodioxane and methylisothiazolone, with ahealth rating indicating that short exposure could cause serioustemporary or residual injury and a flammability rating indicating thatit could be ignited under most ambient conditions. Another preservativeis the StabilZyme Select® Conjugate Stabilizer (SurModics, Inc., EdenPrairie, Minn.) which is an aqueous protein-containing mixture preservedwith methylisothiazolone and bromonitrodioxane. Yet another line ofpreservative is ProClin (Supelco Inc., Bellefonte, Pa.) which utilizes5-chloro-2-methyl-4-isothiazolin3-one and 2-methyl-4-isothiazolin-3-one.Isothiazolone and its derivatives are corrosive to the eyes potentiallycausing permanent irreversible injury, can cause skin burns orirritation, and are considered toxic to fish and wildlife if permittedto enter the water supply. Bromonitrodioxane is a formaldehyde releaser,and since formaldehyde is carcinogenic and highly flammable in liquidand gaseous forms, bromonitrodioxane is an unfavorable candidate as apreservative for samples processed in high volume. Consequently, thereis a need for an environmentally friendly system which can preserve abiological reagent while maintaining its chemical and physicalproperties.

Preservatives are often added to therapeutics to increase shelf-life andto reduce the possibility of microbial contamination. As in the case ofbiological reagents, many therapeutics are packaged in multiple-entrycontainers at volumes which allow the extraction of small aliquots overtime. For example, vaccines are routinely provided in multiple entrycontainers, and for several decades, thimerosal, a mercury-basedpreservative, has been used in vaccines to prevent contamination andother biologics in multidose containers. The Food and DrugAdministration (FDA) has undertaken a review of drugs containingmercury-based preservatives, including thimerosal, in an effort toreduce the concentration of mercury in vaccines and to find alternativepreservative formulations that do not contain mercury. (“Recommendationsregarding the use of vaccines that contain thimerosal as apreservative,” MMWR 48:996-998 (Nov. 5, 1999)) Therefore, there is aneed to provide safe, effective preservatives for therapeutics whichreduce the risk of microbial contamination as well as potential healthproblems associated with exposure to mercury.

Microbial contamination can also lead to the chemical and/or physicaldegradation of personal care products such as cosmetics, hand cleansers,lotions, and shampoos. Moreover, contaminated products routinely exhibitdiminished performance and contribute to the spread of infection tousers.

Work surfaces and equipment in hospitals and laboratories are highlysusceptible to microbial contamination. Likewise, surfaces andappliances found in kitchens and bathrooms of households, restaurants,groceries, catering establishments and the like are routinely exposed tomicrobial contamination. There is a continuing need for environmentallysafe, effective sanitizing products capable of reducing the microbialbioburden in these areas as well.

SUMMARY OF THE INVENTION

In one aspect, the invention is a system for preserving a sample whichmay contain microorganisms, the system including an effective amount ofa composition comprising a biguanide and at least one otherantimicrobial agent, and the composition being cidal to themicroorganisms when present in the sample and containing noantimicrobial additive having a National Fire Protection Associationhealth hazard rating higher than moderate. In one embodiment, thebiguanide utilized in the system is chlorhexidine. In anotherembodiment, the composition comprises a biguanide and at least one otherantimicrobial agent comprising a compound that reduces the selectivepermeability of the cell membrane of the microorganisms. In anotherembodiment, the composition comprises chlorhexidine and at least oneother antimicrobial agent comprising a compound that reduces theselective permeability of the cell membrane of the microorganisms. Inanother embodiment, the composition comprises a biguanide and anaromatic alcohol. In another embodiment, the composition compriseschlorhexidine and an aromatic alcohol. In another embodiment, thecomposition comprises a biguanide and 2-phenyl ethanol. In anotherembodiment, the composition comprises chlorhexidine and 2-phenylethanol. In another embodiment, the composition comprises a biguanide anaromatic alcohol, and a terpenoid. In another embodiment, thecomposition comprises chlorhexidine, an aromatic alcohol, and aterpenoid. In another embodiment, the composition comprises a biguanide,2-phenyl ethanol, and a terpenoid. In another embodiment, thecomposition comprises chlorhexidine, 2-phenyl ethanol, and a terpenoid.In another embodiment, the composition comprises a biguanide, anaromatic alcohol, and isoeugenol. In another embodiment, the compositioncomprises chlorhexidine, an aromatic alcohol, and isoeugenol. In anotherembodiment, the composition comprises a biguanide, 2-phenyl ethanol, andisoeugenol. In another embodiment, the composition compriseschlorhexidine, 2-phenyl ethanol, and isoeugenol. In another embodiment,the composition comprises a biguanide and a propionate. In anotherembodiment, the composition comprises chlorhexidine and a propionate. Inanother embodiment, the composition comprises a biguanide and sodiumpropionate. In another embodiment, the composition compriseschlorhexidine and sodium propionate. In another embodiment, thecomposition comprises a biguanide, a propionate, and aparahydroxybenzoate. In another embodiment, the composition compriseschlorhexidine, a propionate, and a parahydroxybenzoate. In anotherembodiment, the composition comprises a biguanide, sodium propionate,and parahydroxybenzoate. In another embodiment, the compositioncomprises chlorhexidine, sodium propionate, and parahydroxybenzoate. Inanother embodiment, the composition comprises a biguanide, a propionate,and ethyl parahydroxybenzoate. In another embodiment, the compositioncomprises chlorhexidine, a propionate, and ethyl parahydroxybenzoate. Inanother embodiment, the composition comprises a biguanide, sodiumpropionate, and ethyl parahydroxybenzoate. In another embodiment, thecomposition comprises chlorhexidine, sodium propionate, and ethylparahydroxybenzoate. In another embodiment, the-composition comprises abiguanide and boric acid or a boric acid derivative. In anotherembodiment, the composition comprises chlorhexidine and boric acid or aboric acid derivative. In another embodiment, the composition comprisesa biguanide, a propionate, and boric acid or a boric acid derivative. Inanother embodiment, the composition comprises chlorhexidine, apropionate, and boric acid or a boric acid derivative. In anotherembodiment, the composition comprises a biguanide, sodium propionate,and boric acid or a boric acid derivative. In another embodiment, thecomposition comprises chlorhexidine, sodium propionate, and boric acidor a boric acid derivative. The system is useful for samples selectedfrom patient specimens, bodily fluids, reagents, therapeutics, personalcare products and sanitizable surface.

In another aspect, the invention is a device comprising an accessible,sealable enclosure for containing and preserving a sample which maycontain microorganisms, the enclosure containing a composition free oftoxins including mercury, mercury containing compounds, formaldehyde,formaldehyde-releasing compounds, and azides which are unsuitable foruse in high volume processing, and comprising a biguanide and at leastone other antimicrobial agent, the composition being cidal tomicroorganisms when present in the sample. In one embodiment, thebiguanide utilized in the system is chlorhexidine. In anotherembodiment, the composition comprises a biguanide and at least one otherantimicrobial agent comprising a compound that reduces the selectivepermeability of the cell membrane of the microorganisms. In anotherembodiment, the composition comprises chlorhexidine and at least oneother antimicrobial agent comprising a compound that reduces theselective permeability of the cell membrane of the microorganisms. Inanother embodiment, the composition comprises a biguanide and anaromatic alcohol. In another embodiment, the composition compriseschlorhexidine and an aromatic alcohol. In another embodiment, thecomposition comprises a biguanide and 2-phenyl ethanol. In anotherembodiment, the composition comprises chlorhexidine and 2-phenylethanol. In another embodiment, the composition comprises a biguanide anaromatic alcohol, and a terpenoid. In another embodiment, thecomposition comprises chlorhexidine, an aromatic alcohol, and aterpenoid. In another embodiment, the composition comprises a biguanide,2-phenyl ethanol, and a terpenoid. In another embodiment, thecomposition comprises chlorhexidine, 2-phenyl ethanol, and a terpenoid.In another embodiment, the composition comprises a biguanide, anaromatic alcohol, and isoeugenol. In another embodiment, the compositioncomprises chlorhexidine, an aromatic alcohol, and isoeugenol. In anotherembodiment, the composition comprises a biguanide, 2-phenyl ethanol, andisoeugenol. In another embodiment, the composition compriseschlorhexidine, 2-phenyl ethanol, and isoeugenol. In another embodiment,the composition comprises a biguanide and a propionate. In anotherembodiment, the composition comprises chlorhexidine and a propionate. Inanother embodiment, the composition comprises a biguanide and sodiumpropionate. In another embodiment, the composition compriseschlorhexidine and sodium propionate. In another embodiment, thecomposition comprises a biguanide, a propionate, and aparahydroxybenzoate. In another embodiment, the composition compriseschlorhexidine, a propionate, and a parahydroxybenzoate. In anotherembodiment, the composition comprises a biguanide, sodium propionate,and parahydroxybenzoate. In another embodiment, the compositioncomprises chlorhexidine, sodium propionate, and parahydroxybenzoate. Inanother embodiment, the composition comprises a biguanide, a propionate,and ethyl parahydroxybenzoate. In another embodiment, the compositioncomprises chlorhexidine, a propionate, and ethyl parahydroxybenzoate. Inanother embodiment, the composition comprises a biguanide, sodiumpropionate, and ethyl parahydroxybenzoate. In another embodiment, thecomposition comprises chlorhexidine, sodium propionate, and ethylparahydroxybenzoate. In another embodiment, the composition comprises abiguanide and boric acid or a boric acid derivative. In anotherembodiment, the composition comprises chlorhexidine and boric acid or aboric acid derivative. In another embodiment, the composition comprisesa biguanide, a propionate, and boric acid or a boric acid derivative. Inanother embodiment, the composition comprises chlorhexidine, apropionate, and boric acid or a boric acid derivative. In anotherembodiment, the composition comprises a biguanide, sodium propionate,and boric acid or a boric acid derivative. In another embodiment, thecomposition comprises chlorhexidine, sodium propionate, and boric acidor a boric acid derivative. The device is useful for samples selectedfrom patient specimens, bodily fluids, reagents, therapeutics, andpersonal care products.

In another aspect, the invention is an improved method for preserving asample which may contain microorganisms, the improvement comprisingpreserving the chemical and physical properties of the sample by mixingthe sample with an effective amount of a composition comprising abiguanide and at least one other antimicrobial agent, wherein thecomposition is cidal to microorganisms when present in the sample. Inone embodiment, the biguanide utilized in the system is chlorhexidine.In another embodiment, the composition comprises a biguanide and atleast one other antimicrobial agent comprising a compound that reducesthe selective permeability of the cell membrane of the microorganisms.In another embodiment, the composition comprises chlorhexidine and atleast one other antimicrobial agent comprising a compound that reducesthe selective permeability of the cell membrane of the microorganisms.In another embodiment, the composition comprises a biguanide and anaromatic alcohol. In another embodiment, the composition compriseschlorhexidine and an aromatic alcohol. In another embodiment, thecomposition comprises a biguanide and 2-phenyl ethanol. In anotherembodiment, the composition comprises chlorhexidine and 2-phenylethanol. In another embodiment, the composition comprises a biguanide anaromatic alcohol, and a terpenoid. In another embodiment, thecomposition comprises chlorhexidine, an aromatic alcohol, and aterpenoid. In another embodiment, the composition comprises a biguanide,2-phenyl ethanol, and a terpenoid. In another embodiment, thecomposition comprises chlorhexidine, 2-phenyl ethanol, and a terpenoid.In another embodiment, the composition comprises a biguanide, anaromatic alcohol, and isoeugenol. In another embodiment, the compositioncomprises chlorhexidine, an aromatic alcohol, and isoeugenol. In anotherembodiment, the composition comprises a biguanide, 2-phenyl ethanol, andisoeugenol. In another embodiment, the composition compriseschlorhexidine, 2-phenyl ethanol, and isoeugenol. In another embodiment,the composition comprises a biguanide and a propionate. In anotherembodiment, the composition comprises chlorhexidine and a propionate. Inanother embodiment, the composition comprises a biguanide and sodiumpropionate. In another embodiment, the composition compriseschlorhexidine and sodium propionate. In another embodiment, thecomposition comprises a biguanide, a propionate, and aparahydroxybenzoate. In another embodiment, the composition compriseschlorhexidine, a propionate, and a parahydroxybenzoate. In anotherembodiment, the composition comprises a biguanide, sodium propionate,and parahydroxybenzoate. In another embodiment, the compositioncomprises chlorhexidine, sodium propionate, and parahydroxybenzoate. Inanother embodiment, the composition comprises a biguanide, a propionate,and ethyl parahydroxybenzoate. In another embodiment, the compositioncomprises chlorhexidine, a propionate, and ethyl parahydroxybenzoate. Inanother embodiment, the composition comprises a biguanide, sodiumpropionate, and ethyl parahydroxybenzoate. In another embodiment, thecomposition comprises chlorhexidine, sodium propionate, and ethylparahydroxybenzoate. In another embodiment, the composition comprises abiguanide and boric acid or a boric acid derivative. In anotherembodiment, the composition comprises chlorhexidine and boric acid or aboric acid derivative. In another embodiment, the composition comprisesa biguanide, a propionate, and boric acid or a boric acid derivative. Inanother embodiment, the composition comprises chlorhexidine, apropionate, and boric acid or a boric acid derivative. In anotherembodiment, the composition comprises a biguanide, sodium propionate,and boric acid or a boric acid derivative. In another embodiment, thecomposition comprises chlorhexidine, sodium propionate, and boric acidor a boric acid derivative. The device is useful for samples selectedfrom patient specimens, bodily fluids, reagents, therapeutics, andpersonal care products.

DETAILED DESCRIPTION

A transport/preservative system has been found which provides safe,effective transport for patient specimens and bodily fluids, andfunctions as a preservative for biological reagents, therapeutics, andpersonal care products. The transport/preservative formulations can alsobe used as safe, effective sanitizers on surfaces, equipment, andappliances.

In describing the features of the transport/preservative system of thepresent invention, the following terms are defined as given below. Theterm “cidal” is defined as having antimicrobial activity against grampositive and gram negative bacteria and yeasts, wherein the effectiveantimicrobial strength is sufficient to cause a reduction of at least2-3 logs in organism count within 24 hours or to cause sufficient damageto the microorganisms present so that their metabolism is arrested to alevel at which the microorganisms cannot alter the chemical or physicalproperties of the sample. Unless otherwise stated, the term “sample”refers to either a diagnostic or laboratory sample, bodily fluid,biological reagent, personal care product, or therapeutic. The term“therapeutic” includes topically, subcutaneously, intramuscularly,orally, mucosally, and unguinally administered pharmaceuticals,nutraceuticals, and over-the-counter preparations.

The transport/preservative system of the present invention utilizesantimicrobial agents having low environmental impact to synergisticallyprovide microbiocidal activity. The transport/preservative system of thepresent invention provides cidal, not static, antimicrobial activityagainst the majority of microorganisms commonly found in diagnosticsamples, biological reagents, personal care products, or therapeutics,thereby effectively reducing the microbial count to levels which willnot cause chemical or physical degradation for extended periods of timeat room temperature. Use of the transport/preservative system of thepresent invention does not alter the chemical properties of interest ofthe sample. For example, the amount of glucose, ketone, protein,urobilinogen, nitrite, and blood in a patient urine sample can bemaintained for extended periods of time. Likewise, thetransport/preservative system of the present invention does not alterthe physical properties of interest of the sample, e.g., presence ofleukocytes, cast cells or red blood cells, specific gravity, color, pH,or buffering capacity of a sample. If desired, the morphologicalintegrity of killed microorganisms in samples can be preserved for over72 hours, thus allowing meaningful microscopic analysis. The majorcomponents of the transport/preservative formulations of the presentinvention which provide its antimicrobial action are shelf-stable andhave a moderate to low NFPA hazard rating indicative of environmentalsafety. The transport/preservative system of the present inventionutilizes combinations of at least two antimicrobial components toachieve cidal action against microorganisms while maintaining thechemical and physical integrity of the sample, thus reducing thepossibility of the development of resistance in the microorganisms ofinterest to one particular antimicrobial component. Additionally, theantimicrobial components of the transport/preservative system actsynergistically to permit the use of lower effective concentrations ofeach component, thereby reducing any possible deleterious effects ofindividual components on the chemical and physical properties of thesample as well as limiting the exposure of patients, healthcareprofessionals, laboratory personnel, and consumers to the components;The antimicrobial action of the transport/preservative formulations isalso sufficient to sanitize surfaces, containers, equipment, appliancesand the like. The variety of transport/preservative formulationspresented herein include formulations which are compatible with glass,plastic, rubber, and metal, thus permitting use of thetransport/preservative system with a wide range of surfaces, containers,device formats, and instrumentation.

The transport/preservative system of the present invention comprises atleast two antimicrobial components. By using more than one antimicrobialcomponent, the transport/preservative system increases the probabilityof controlling the growth and/or metabolism of potentially resistantmicroorganisms. The effective combination of antimicrobial componentsmust be cidal. For patient samples which require microscopicexamination, e.g., urine specimens, the antimicrobial components of thetransport/preservative system of the present invention can be selectedto preserve gross cellular morphology.

The antimicrobial components of the present invention must be solubleand chemically stable at room temperature in aqueous solutions ateffective concentrations. Another characteristic of the antimicrobialcomponents is that, at effective concentrations, they cannot possesssufficient acidity, basicity, or buffering capacity so as to alter thepH of the sample. The antimicrobial components must be stable andeffective through a broad pH range, i.e, from about pH 4 to about pH 8.For samples where specific gravity is considered an important parameter,e.g., urine samples, the specific gravity of the antimicrobialcomponents in aqueous solution must be sufficiently similar to water soas to not change the specific gravity of the sample.

Antimicrobial biguanides are one important class of compounds used inthe present invention. Among the useful biguanides are chlorhexidine andits derivatives (e.g., chlorhexidine gluconate), the alexidine group,and polymeric biguanides (e.g., polyhexamethylene biguanides). Thepreferred biguanides are chlorhexidine and its derivatives. It isunderstood that similar biguanides with bromide and/or iodide ionssubstituted for the chloride ions can be used in the present invention.

Antimicrobial agents which damage the cell membrane of microorganismsand subsequently reduces or destroys the cell membrane's selectivepermeability are also important components of the present invention.These include but are not limited to antimicrobial aromatic alcohols,terpenoids, parahydroxybenzoate esters, deoxycholates, taurocholates,and detergents/surfactants. Suitable antimicrobial aromatic alcoholsinclude but are not limited to phenylethyl alcohol, benzyl alcohol, andphenoxyethyl alcohol. Preferred terpenoids include but are not limitedto isoeugenol, isohexanol, and isooctanol. Suitable parahydroxybenzoateesters include but are not limited to alkyl esters such as methyl-,ethyl-, propyl-, and butyl-parahydroxybenzoates as well as the aromaticbenzylparahydroxybenzoate. Preferred detergents/surfactants arelipid-active such as octoxynol (Triton-X).

Antimicrobial organic acids are another important class of antimicrobialcomponents of the present invention. Preferred organic acids include butare not limited to acetic, propionic, benzoic, citrate, and sorbic acidand their monvalent salts. In urine transport, ammonium salts are notuseful due to their interference in routine diagnostics tests. Mostpreferred is sodium propionate.

Boric acid and its derivatives are other important antimicrobialcomponents of the present invention. Unlike commercially availablespecimen transport systems using boric acid to provide microbial stasis,the transport/preservative system of the present invention uses boricacid in combination with other antimicrobial components to providemicrobial cidal action.

Optionally, the transport/preservative system of the present inventioncan comprise fragrance components. Preferably, the fragrance componentsare antimicrobial. Preferred fragrance components include but are notlimited to isoeugenol, ethyl vanillin, and pinacol.

A preferred stabilizing, transport/preservative formulation of thepresent invention comprises an antimicrobial aromatic alcohol, an alkylguaiacol, and a biguanide. One example of this formulation, hereinafterreferred to as “Chemistat I,” comprises 2-phenyl ethanol, isoeugenol,and chlorhexidine. Most preferably, Chemistat I formulations comprise2-phenyl ethanol from about 0 μl/ml to about 2.5 μl/ml, isoeugenol fromabout 0.2 μl/ml to about 1.5 μl/ml, and chlorhexidine from about 0.01mg/ml to about 0.1 mg/ml, and any combination thereof. Theseconcentrations and subsequent concentrations for thetransport/preservative system reported herein are the finalconcentration after the sample is added. To preserve urine specimens,the Chemistat I formulation preferably comprises 2-phenyl ethanol atabout 1.8 μl/ml, isoeugenol at about 0.2 μl/ml, and chlorhexidine atabout 0.02 mg/ml. The Chemistat I formulations are in liquid form, and2-phenyl ethanol, isoeugenol, and chlorhexidine are all activeingredients. Additionally, 2-phenyl ethanol and isoeugenol provide asynthetic rose scent and a spicy fragrance, respectively, to mask sampleodor.

EXAMPLE 1 Effectiveness of Chemistat I Formulations

Chemistat I formulations were shown to preserve urine samples containingsignificant microbial contamination and at the same time maintain thechemical and physical properties of the urine samples.

Antimicrobial activity for each active ingredient in the Chemistat Iformulations was first determined for representative organisms listed inTable I. A culture for each organism of interest on blood agar wasincubated at 36° C. overnight. About two to three colonies of theorganism were transferred via sterile swab from the blood agar cultureinto a 5 ml Mueller-Hinton broth culture tube, which was then incubatedon a rotator for about two to three hours at 36° C. Using aspectrophotometer set at a wavelength of 630 nm, the turbidity of brothculture was adjusted by the addition of Mueller-Hinton broth to anoptical density equivalent to approximately 1×10⁸ colony forming units(cfu)/ml to form an inoculum solution. An aliquot of the inoculumsolution was plated onto blood agar to determine the actual controlorganism count. For each component listed in Table II, the component wasadded to filter-sterilized pooled normal urine at the concentrationgiven in Table II to form a test sample. The test sample was theninoculated with an aliquot of the inoculum solution providing a finalorganism count of approximately 1×10⁶ cfu/ml of test sample. Immediatelyafter thorough mixing, an aliquot of the test sample was removed andserially diluted in PBS, and serial dilutions were plated onto bloodagar to determine the Time Zero organism count. The inoculated plateswere incubated at 36° C., and organism counts were recorded after 18-24hours. After incubating the test sample for 24 hours at roomtemperature, an aliquot of the test sample was again removed, seriallydiluted in PBS and plated onto blood agar. After incubating for 18-24hours at 36° C., organism counts were obtained. The organism countobtained after 24 hours incubation at room temperature was divided bythe control organism count to provide the survival value of eachorganism as recorded in Table II. With respect to changes in microbialcount, a survival value of 1 represents no change; a survival value of0.1 indicates a 1-log decrease; a survival value of 10 indicates a 1-logincrease; and a survival value of 0.000 represents a decrease of greaterthan or equal to 3-logs. In comparison, the lower the survival value,the greater the component's capacity for controlling organism growth.

As shown in Table II, 2-phenyl ethanol alone exhibited cidal activityagainst Escherichia coli at 4 μl/ml; Pseudomonas aeruginosa, at 5 μl/ml;Enterobacter aerogenes and Candida albicans, at 10 μl/ml; but was notcidal against Enterococcus faecalis at up to 10 μl/ml. Further, 2-phenylethanol produced a strong unpleasant odor when combined with urine atconcentrations of 4-10 μl/ml, making it undesirable for use as a urinepreservative at these concentrations. Isoeugenol showed cidal activityagainst Escherichia coli at 1.5 μl/ml; Candida albicans and Enterobacteraerogenes, at 2.5 μl/ml; but was not cidal against Enterococcus faecalisat up to 4.6 μl/ml. Chlorhexidine had cidal activity against Escherichiacoli and Candida albicans at 0.0075 mg/ml; Enterobacter aerogenes, at0.01 mg/ml; Proteus vulgaris and Klebsiella pneumoniae, at 0.02 mg/ml;showed variable cidal activity against Enterococcus faecalis at 0.05-0.8mg/ml; and was not cidal against Pseudomonas aeruginosa at up to 0.05mg/ml. In summary, the three active ingredients each had cidal activityagainst some but not all of the microorganisms of interest. Cidalantimicrobial action for many organisms of interest with isoeugenolalone required a concentration greater than 1.5 μg/ml; cidal action with2-phenyl ethanol alone required a concentration greater than 2.5 μl/ml;cidal action with chlorhexidine required a concentration of greater thanor equal to 0.01 mg/ml.

TABLE I List of Organisms Used Organism Name Code Ref. NumberAcinetobacter calcoaceticus acca1 14290^(a) acca2 23055^(a) Alcaligenesfaecalis alfa1  8750^(a) alfa2 35655^(a) Candida albicans caal114053^(a) caal2 60193^(a) Citrobacter diversus cidi1 29225^(a) cidi23220-2^(a) Citrobacter freundii cifr1 33128^(a) cifr2  699^(b)Enterobacter aerogenes enae1 13048^(a) enae2  2490^(b) Enterobactercloacae encl1 29006^(a) encl2 3118-1^(b) Enterococcus faecalis enfa129212^(a) enfa2 49477^(a) Escherichia coli esco1 25922^(a) esco211303^(a) esco3 29194^(a) esco4 8739-1^(b) Klebsiella oxytoca klox143165^(a) klox2 33496^(a) Klebsiella pneumoniae klpn1 33495^(a) klpn213883^(a) Morganella morganii momo1 25830^(a) momo2 29853^(a) Proteusmirabilis prmi1  7002^(a) prmi2 12453^(a) Proteus vulgaris prvu113315^(a) prvu2 49132^(a) Providencia stuartii prst1 29914^(a) prst249809^(a) Pseudomonas aeruginosa psae1 27853^(a) psae2 10145^(a)Serratia marcescens sema1  9103^(a) sema2  3155^(b) Staphylococcusaureus stau1 25923^(a) stau2 33591^(a) Streptococcus agalactiae stag1 624^(a) stag2 13813^(a) ^(a)Strains obtained from the American TypeCulture Collection (ATCC). ^(b)Strains obtained from patient samples andidentified by routine clinical microbiology tests.

TABLE II Antimicrobial Activity for Individual Chemistat I ComponentsSurvival Values^(a) after 24 hours at Room Temperature forRepresentative Organisms^(b) Component esco psae prvu enfa caal klpnenae 2-Phenyl ethanol (1 μl/ml) tntc 4.819 tntc tntc 2-Phenyl ethanol(1.8 μl/ml) tntc tntc tntc tntc tntc tntc 2-Phenyl ethanol (2.5 μl/ml)0.245 0.199 tntc 0.092 2-Phenyl ethanol (4 μl/ml)^(c) 0.000 0.019 tntc1.081 0.244 2-Phenyl ethanol (5 μl/ml)^(c) 0.000 tntc 0.440 2-Phenylethanol (10 μl/ml)^(c) 0.000 0.000 1.495 0.000 0.000 Chlorhexidine(0.005 mg/ml) 0.003 tntc 1.115 5.308 tntc Chlorhexidine (0.0075 mg/ml)0.000 4.049 0.598 0.000 6.098 Chlorhexidine (0.01 mg/ml) 0.000 0.2770.520 0.000 Chlorhexidine (0.02 mg/ml) 0.000 0.275 0.000 0.179Chlorhexidine (0.02 mg/ml) 0.000 0.080 0.000 0.141 0.000 0.000Chlorhexidine (0.05 mg/ml) 0.000 0.046 0.000 0.000 0.000 Chlorhexidine(0.1 mg/ml) 0.000 0.561 0.000 Chlorhexidine (0.8 mg/ml)^(d) 0.000 0.0000.000 Isoeugenol (0.2 μl/ml) tntc tntc tntc tntc 0.160 tntc Isoeugenol(0.5 μl/ml) tntc tntc 2.786 Isoeugenol (1.5 μl/ml) 0.000 tntc tntc 0.152tntc Isoeugenol (2.5 μl/ml)^(c) 0.000 0.398 0.750 0.000 0.000 Isoeugenol(4.6 μl/ml)^(c) 0.000 0.460 0.000 ^(a)Numbers indicate survival values(number of organisms recovered after 24 hours incubation at roomtemperature divided by the initial control count; tntc = organisms at 24hours too numerous to count. ^(b)Organism Key: esco = Escherichia coli;psae = Pseudomonas aeruginosa; prvu = Proteus vulgaris; enfa =Enterococcus faecalis; caal = Candida albicans; klpn = Klebsiellapneumoniae, enae = Enterobacter aerogenes. ^(c)Strong unpleasant odor.^(d)Produced a false positive protein upon urinalysis.

As shown in Table III, the components of the Chemistat I formulations ofthe present invention act synergistically with each other, in that theeffective concentration of each individual component of the formulationnecessary for cidal action against the microorganisms of interest isless than the effective concentration of each component testedseparately, and also a wider range of microorganisms are killed with theformulation. For example, neither isoeugenol at 1.5 μl/ml norchlorhexidine at 0.005 mg/ml alone are cidal against Candida albicans,Pseudomonas aeruginosa, or Enterobacter aerogenes, but the combinationis cidal against Candida albicans and Enterobacter aerogenes andinhibitory against Pseudomonas aeruginosa. Isoeugenol at 0.2 μl/ml wasnot cidal against any of the organisms of interest and chlorhexidine at0.02 mg/ml was not cidal against Pseudomonas aeruginosa and Enterococcusfaecalis; however, the combination was cidal against all organisms ofinterest given in Table III. Neither isoeugenol at 0.2 μl/ml nor2-phenyl ethanol at 1.8 μl/ml had any effect against Escherichia coliwhen used alone, but when used together, they inhibited Escherichiacoli. Chlorhexidine at 0.02 mg/ml was not cidal against Pseudomonasaeruginosa and Enterococcus faecalis, and 2-phenyl ethanol at 1.8 μl/mldid not inhibit any organism of interest. However, the combination ofchlorhexidine (0.02 mg/ml) and 2-phenyl ethanol (1.8 μl/ml) was cidalagainst all organisms of interest in Table III. Extensive studies usingall of the organisms listed in Table I showed that Chemistat Iformulations stabilize urine samples initially containing approximately1×10⁶ cfu/ml for a period of seven days at room temperature (22-25° C.).(Data not shown) It is anticipated that other fragrances such as ethylvanillin (2 mg/ml) or pinacol (10 mg/ml) can be added to Chemistat Iformulations, e.g., a combination of isoeugenol (0.2 μl/ml) andchlorhexidine (0.02 mg/ml) to produce a cidal transport/preservativesystem.

TABLE III Antimicrobial Activity for Chemistat I Component CombinationsSurvival Values^(a) after 24 hours at Room Temperature forRepresentative Organisms^(b) Component Combinations esco psae prvu enfacaal klpn enae Isoeugenol (1.5 μl/ml); 0.000 0.032 0.780 0.000 0.000chlorhexidine (0.005 mg/ml) 2-Phenyl ethanol (5 μl/ml); 0.000 0.0000.000 0.000 chlorhexidine (0.1 mg/ml)^(c) 2-Phenyl ethanol (5 μl/ml);0.000 0.000 0.023 0.000 0.000 chlorhexidine (0.01 mg/ml)^(c) 2-Phenylethanol (2.5 μl/ml); 0.000 0.136 0.730 0.757 0.000 chlorhexidine (0.005mg/ml) 2-Phenyl ethanol (1.25 μl/ml); 0.000 tntc 1.157 2.748 0.988chlorhexidine (0.0025 mg/ml) 2-Phenyl ethanol (4 μl/ml); 0.000 0.0000.128 0.000 0.000 chlorhexidine (0.0075 mg/ml)^(c) 2-Phenyl ethanol (1.8μl/ml); 0.147 tntc tntc 0.306 tntc isoeugenol (0.2 μl/ml) 2-Phenylethanol (1.8 μl/ml); 0.000 0.000 0.214 0.121 0.000 isoeugenol (0.2μl/ml); chlorhexidine (0.005 mg/ml) 2-Phenyl ethanol (1.8 μl/ml); 0.0000.000 0.075 0.000 0.000 isoeugenol (0.2 μl/ml); chlorhexidine (0.01mg/ml) 2-Phenyl ethanol (3.8 μl/ml); 0.000 0.105 tntc 0.758 0.000isoeugenol (0.2 μl/ml)^(c) 2-Phenyl ethanol (3.8 μl/ml); 0.000 0.0000.000 0.185 0.000 isoeugenol (0.2 μl/ml); chlorhexidine (0.005mg/ml)^(c) 2-Phenyl ethanol (3.8 μl/ml); 0.000 0.000 0.000 0.000 0.094isoeugenol (0.2 μl/ml); chlorhexidine (0.01 mg/ml)^(c) 2-Phenyl ethanol(1.8 μl/ml); 0.487 tntc 1.315 tntc 0.136 tntc isoeugenol (0.2 μl/ml)2-Phenyl ethanol (1.8 μl/ml); 0.000 0.000 0.000 0.000 0.000 0.000chlorhexidine (0.02 mg/ml) Isoeugenol (0.2 μl/ml); 0.000 0.000 0.0000.000 0.000 0.000 chlorhexidine (0.02 mg/ml) 2-Phenyl ethanol (1.8μl/ml); 0.000 0.000 0.000 0.000 0.000 0.000 chlorhexidine (0.02 mg/ml);isoeugenol (0.2 μl/ml) ^(a)Numbers indicate survival values (number oforganisms recovered after 24 hours incubation at room temperaturedivided by the initial control count; tntc = organisms at 24 hours toonumerous to count. ^(b)Organism Key: esco = Escherichia coli; psae =Pseudomonas aeruginosa; prvu = Proteus vulgaris; enfa = Enterococcusfaecalis; caal = Candida albicans; klpn = Klebsiella pneumoniae; enae =Enterobacter aerogenes. ^(c)Strong unpleasant odor.

With respect to urinalysis, the components of the Chemistat Iformulations at their preferred concentrations did not affect anyurinalysis parameter or microbial morphology when normal pooled urinewas inoculated with microorganisms of interest at 1×10⁶ cfu/ml. However,chlorhexidine at higher concentrations of greater than or equal to 0.8mg/ml caused false positive protein readings.

Because of possible deleterious interactions of the Chemistat Iformulation with plastics and possibly with the rubber stoppers and/orsilicone commonly used in packaging, transport and/or processingcontainers, stabilizing transport/preservative formulations in powder,liquid, or lyophilized form which are inert to plastics, rubber, andsilicone, hereinafter referred to as “Chemistat II”, have been found,comprising an antimicrobial organic acid, an antimicrobial agent whichattacks or disrupts the lipid membrane of microorganisms of interest,and antimicrobial biguanide. Preferably, Chemistat II comprises anantimicrobial proprionate, parahydroxy benzoate, and a biguanide. Mostpreferably, Chemistat II comprises sodium propionate, ethylparahydroxybenzoate, and chlorhexidine. Preferred Chemistat IIformulations comprise from about 1 mg/ml to about 10 mg/ml sodiumproprionate, from about 0.1 mg/ml to about 1 mg/ml ethylparahydroxybenzoate, and from about 0.01 mg/ml to about 0.1 mg/mlchlorhexidine, and any combination thereof. For preservation of urinespecimens, a preferred Chemistat II formulation comprises 6 mg/ml sodiumproprionate, 0.5 mg/ml ethyl parahydroxybenzoate, and 0.025 mg/mlchlorhexidine. Another preferred Chemistat II formulation for urinespecimens comprises 6.25 mg/ml sodium propionate, 0.25 mg/ml ethylparahydroxybenzoate, and 0.0275 mg/ml chlorhexidine.

EXAMPLE 2 Effectiveness of Chemistat II Formulations

Chemistat II formulations were shown to preserve urine samplescontaining significant microbial contamination and at the same timemaintain the chemical and physical properties of the urine samples.

The antimicrobial results for the individual components are given inTable IV. As previously discussed, chlorhexidine alone had cidalactivity against Escherichia coli and Candida albicans at 0.0075 mg/ml;Enterobacter aerogenes, at 0.01 mg/ml; Proteus vulgaris and Klebsiellapneumoniae, at 0.02 mg/ml; showed variable cidal activity againstEnterococcus faecalis at 0.05-0.8 mg/ml; and was not cidal againstPseudomonas aeruginosa at up to 0.05 mg/ml. Ethyl parahydroxybenzoatewas cidal against Escherichia coli at 1 mg/ml; inhibitory againstCandida albicans and Enterobacter aerogenes, at 0.5 mg/ml and 1 mg/ml,respectively; and ineffective against Pseudomonas aeruginosa, Proteusvulgaris, Enterococcus faecalis, and Klebsiella pneumoniae at up to 1mg/ml. Sodium propionate was inhibitory against Pseudomonas aeruginosaand Enterococcus faecalis at 10 mg/ml, but was ineffective againstEscherichia coli, Proteus vulgaris, Candida albicans, Klebsiellapneumoniae, and Enterobacter aerogenes at up to 10 mg/ml. Thus, thecomponents of the Chemistat II formulations when used alone were unableto control the growth of the microorganisms of interest.

TABLE IV Antimicrobial Activity for Individual Chemistat II ComponentsSurvival Values^(a) after 24 hours at Room Temperature forRepresentative Organisms^(b) Component esco psae prvu enfa caal klpnenae Chlorhexidine (0.005 mg/ml) 0.003 tntc 1.115 5.308 tntcChlorhexidine (0.0075 mg/ml) 0.000 4.049 0.598 0.000 6.098 Chlorhexidine(0.01 mg/ml) 0.000 0.277 0.520 0.000 Chlorhexidine (0.02 mg/ml) 0.0000.275 0.000 0.179 Chlorhexidine (0.02 mg/ml) 0.000 0.080 0.000 0.1410.000 0.000 Chlorhexidine (0.05 mg/ml) 0.000 0.046 0.000 0.000 0.000Chlorhexidine (0.1 mg/ml) 0.000 0.561 0.000 Chlorhexidine (0.8mg/ml)^(c) 0.000 0.000 0.000 Ethyl parahydroxybenzoate tntc tntc tntctntc 0.362 tntc (0.5 mg/ml)^(d) Ethyl parahydroxybenzoate 0.000 tntctntc 0.208 (1 mg/ml)^(d) Sodium propionate (5 mg/ml) tntc tntc tntc tntctntc tntc Sodium propionate tntc 0.341 2.702 (10 mg/ml) ^(a)Numbersindicate survival values (number of organisms recovered after 24 hoursincubation at room temperature divided by the initial control count;tntc = organisms at 24 hours too numerous to count. ^(b)Organism Key:esco = Escherichia coli; psae = Pseudomonas aeruginosa; prvu = Proteusvulgaris; enfa = Enterococcus faecalis; caal = Candida albicans; klpn =Klebsiella pneumoniae; enae = Enterobacter aerogenes. ^(c)Produced afalse positive protein upon urinalysis. ^(d)Low solubility in urine.

As shown in Table V, combinations of any two of the three Chemistat IIcomponents were not cidal for all microorganisms of interest, inparticular, Pseudomonas aeruginosa and Enterococcus faecalis, at 24hours. However, the combination of chlorhexidine at 0.02 mg/ml, sodiumpropionate at 5 mg/ml, and ethyl parahydroxybenzoate at 0.5 mg/ml wascidal for the organisms of interest. Further, the components of theChemistat II formulations act synergistically with each other, in thatthe effective concentration of each individual component of thecomposition necessary for cidal action against the microorganisms ofinterest is less than the effective concentration of each componenttested separately, and also a wider range of microorganisms are killedwith the composition. For example, chlorhexidine (0.02 mg/ml) aloneinhibits but does not kill P. aeruginosa, and ethyl parahydroxybenzoate(0.5 mg/ml) and sodium propionate (5 mg/ml) together do not affect thisorganism at all. However, when chlorhexidine, ethyl parahydroxybenzoate,and sodium proprionate are added together, the composition of thepresent invention is cidal against P. aeruginosa.

TABLE V Antimicrobial Activity for Chemistat II Component CombinationsSurvival Values^(a) after 24 hours at Room Temperature forRepresentative Organisms^(b) Component Combinations esco psae prvu enfacaal klpn Chlorhexidine (0.02 mg/ml); sodium propionate 0.000 0.0050.000 0.448 (10 mg/ml) Chlorhexidine (0.02 mg/ml); sodium propionate0.000 0.000 0.000 (5 mg/ml); ethyl parahydroxybenzoate (0.5 mg/ml)Chlorhexidine (0.02 mg/ml); sodium propionate 0.026 0.010 (5 mg/ml);ethyl parahydroxybenzoate (0.3 mg/ml) Chlorhexidine (0.02 mg/ml); sodiumpropionate 0.468 0.155 (5 mg/ml); ethyl parahydroxybenzoate (0.1 mg/ml)Ethyl parahydroxybenzoate (0.5 mg/ml); sodium tntc tntc tntc tntc 0.523tntc propionate (5 mg/ml) Ethyl parahydroxybenzoate (0.5 mg/ml); 0.0000.002 0.000 0.000 0.000 0.000 chlorhexidine (0.02 mg/ml) Sodiumpropionate (5 mg/ml); chlorhexidine 0.000 0.326 0.000 0.605 0.000 0.000(0.02 mg/ml) Ethyl parahydroxybenzoate (0.5 mg/ml); 0.000 0.000 0.0000.014 0.000 0.000 chlorhexidine (0.02 mg/ml); sodium proprionate (5mg/ml) ^(a)Numbers indicate survival values (number of organismsrecovered after 24 hours incubation at room temperature divided by theinitial control count; tntc = organisms at 24 hours too numerous tocount. ^(b)Organism Key: esco = Escherichia coli; psae = Pseudomonasaeruginosa; prvu = Proteus vulgaris; enfa = Enterococcus faecalis; caal= Candida albicans; klpn = Klebsiella pneumoniae; enae = Enterobacteraerogenes.

A comparative evaluation was made of the effects of Chemistat II onurinalysis tests. Normal pooled urine samples were added to tubescontaining either boric acid, tartaric acid, Chemistat II medium, ornothing (control) and were then inoculated with microorganisms ofinterest at 1×10⁶ cfu/ml. Urinalysis tests were measured once at TimeZero and, again, after the samples were held for 4, 24, and 48 hours,and 7 days at room temperature. This procedure was also repeated withnormal pooled urine samples to which glucose was added at aconcentration of about 500 mg/dl, i.e., a concentration routinely foundin urine obtained from diabetic patients, so that changes from apositive glucose to a negative glucose over time could be observed.Tables VI and VII summarize comparative results in terms of changes fromTime Zero measurements for Chemistat II, boric acid, and tartaric acidtransport systems without and with glucose added, respectively. Thecomparative tests showed that Chemistat II without added glucosestabilized the urinalysis specimen through 24 hours for all parameters,through 48 hours for all parameters except pH (2° change, e.g., a pHchange from 6.0 to 7.0 or from 5.5 to 6.5), and through 7 days for allparameters except pH (2° change) and specific gravity (2° change, e.g.,a specific gravity change from 1.005 to 1.015 or 1.015 to 1.025).Chemistat II with glucose held all parameters through 48 hours, and onlyone organism showed a pH change (2° change) at 7 days. All of thechanges observed with Chemistat II were not clinically significant,i.e., would not result in a change in the medical interpretation of theurinalysis results. Comparatively, the boric acid and tartaric acidtransport systems without added glucose did not hold specific gravity,pH, nitrites, and blood. Tartaric acid did not hold specific gravity,pH, nitrites, blood, protein, and leukocytes. When glucose was added tothe boric acid transport, glucose, pH, nitrites, and blood were notheld. The tartaric acid transport system with added glucose did not holdglucose, specific gravity, pH, protein, and nitrites. The degree ofchange for the boric acid and tartaric acid transport systems with andwithout glucose added did result in clinically significant changes inthe urinalysis results.

TABLE VI Changes in Urinalysis Results Using Chemistat II, Boric Acid,and Tartaric Acid Transport Systems without Glucose Added Control BoricAcid Tartaric Acid Chemistat II Degree of Change Transport Time^(a)Transport Time^(a) Transport Time^(a) Transport Time^(a) from TimeZero^(b) T4 T24 T48 T7d T4 T24 T48 T7d T4 T24 T48 T7d T4 T24 T48 T7dGlucose^(c)   1° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   2° change 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Bilirubin^(d) 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0   1° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   2°change 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0   3° change 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0   4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Ketone^(e)   1°change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   2° change 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   4° change 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 Specific gravity^(f)   2° change 2 12 13 9 0 00 2 0 1 5 3 0 0 0 1   3° change 0 1 2 5 0 0 0 0 0 0 3 8 0 0 0 0 ≧4°change 0 0 0 0 0 0 0 0 0 0 1 4 0 0 0 0 pH^(g)   2° change 0 2 3 4 0 0 04 0 6 4 3 0 0 1 3   3° change 0 8 11 8 0 0 0 2 0 1 0 0 0 0 0 0 ≧4°change 0 4 15 18 0 0 0 0 0 3 12 18 0 0 0 0 Protein^(h)   1° change 0 1 54 0 0 0 0 0 0 2 3 0 0 0 0   2° change 0 0 3 8 0 0 0 0 0 0 1 6 0 0 0 0  3° change 0 0 1 2 0 0 0 0 0 0 0 1 0 0 0 0   4° change 0 0 1 3 0 0 0 00 0 0 0 0 0 0 0 Urobilinogin^(i)   1° change 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0   2° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   3° change 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0   4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Nitrites^(j)   1° change 1 53 36 22 1 29 34 8 2 46 39 25 0 0 0 0Blood^(k)   1° change 0 2 2 0 0 0 1 2 0 0 0 0 0 0 0 0   2° change 0 0 01 0 0 0 0 0 0 0 1 0 0 0 0   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Leukocytes^(l)   1° change 00 0 3 0 0 0 0 0 1 0 4 0 0 0 0   2° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   4° change 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 ^(a)Transport times: T4 = four hour transport; T24 =twenty-four hour transport; T48 = forty-eight hour transport; T7d =seven day transport; transport time = amount of time elapsed betweenTime Zero when urine sample was inoculated and when sample was testedfor a given parameter. ^(b)Degree of change measured from Time Zero:using the scales given for each urinalysis test result listed below, 1°= a change of one step up or down the scale, e.g., for glucose with 250mg/dl at Time Zero, change to 100 mg/dl or 500 mg/dl; 2° = a change oftwo steps up or down the scale, e.g., for glucose with 250 mg/dl at TimeZero, change to negative or 1000+ mg/dl; 3° = a change of three steps #up or down the scale, e.g., for glucose with 100 mg/dl at Time Zero,change to 1000+ mg/dl; and 4° = a change of four steps up or down thescale, e.g., for glucose with 1000+ mg/dl at Time Zero, change tonegative. ^(c)Possible urinalysis test results for glucose: negative,100, 250, 500, or 1000+ mg/dl. ^(d)Possible urinalysis test results forbilirubin: negative, small, moderate, or large. ^(e)Possible urinalysistest results for ketone: negative, trace, small, moderate, or large.^(f)Possible urinalysis test results for specific gravity: ≧1.005,1.010, 1.015, 1.020, 1.025, or ≧1.030. ^(g)Possible urinalysis testresults for pH ≧5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or ≧9.0. ^(h)Possibleurinalysis test results for protein: negative, trace, 30, 100, 300, or1000+ mg/dl. ^(i)Possible urinalysis test results for urobilinogen: 0.2,1, 2, 4, or 8 mg/dl. ^(j)Possible urinalysis test results for nitrites:negative or positive. ^(k)Possible urinalysis test results for blood:negative, trace, small, moderate or large. ^(l)Possible urinalysis testresults for leukocytes: negative, trace, small, moderate or large.

TABLE VII Changes in Urinalysis Results Using Chemistat II, Boric Acid,and Tartaric Acid Transport Systems with Glucose Added Control BoricAcid Tartaric Acid Chemistat II Degree of Change Transport Time^(a)Transport Time^(a) Transport Time^(a) Transport Time^(a) from TimeZero^(b) T4 T24 T48 T7d T4 T24 T48 T7d T4 T24 T48 T7d T4 T24 T48 T7dGlucose^(c)   1° change 0 4 8 10 0 0 0 3 0 0 1 3 0 0 0 0   2° change 0 07 12 0 0 1 1 0 0 0 0 0 0 0 0   3° change 0 0 2 8 0 0 0 0 0 0 0 0 0 0 0 0  4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Bilirubin^(d)   1° change 00 0 1 0 0 0 0 0 0 0 0 0 0 0 0   2° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   4° change 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 Ketone^(e)   1° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  2° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   3° change 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0   4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Specificgravity^(f)   2° change 1 2 6 15 0 0 0 0 0 0 0 3 0 0 0 0   3° change 0 00 1 0 0 0 0 0 0 0 5 0 0 0 0 ≧4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0pH^(g)   2° change 2 15 12 7 0 0 0 3 0 0 3 13 0 0 0 1   3° change 0 0 147 0 0 0 1 0 0 0 2 0 0 0 0 ≧4° change 0 0 8 27 0 0 0 0 0 0 2 4 0 0 0 0Protein^(h)   1° change 0 0 0 1 0 0 0 0 0 0 1 1 0 0 0 0   2° change 0 00 2 0 0 0 0 0 0 0 2 0 0 0 0   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  4° change 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 Urobilinogin^(i)   1° change0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   2° change 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 Urobilinogin^(i)   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   4°change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nitrites^(j)   1° change 0 2 1 10 4 10 3 0 13 6 1 0 0 0 0 Blood^(k)   1° change 0 0 0 0 0 0 0 2 0 0 0 00 0 0 0   2° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   3° change 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0   4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Leukocytes¹   1° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   2° change 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0   3° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  4° change 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ^(a)Transport times: T4 =four hour transport; T24 = twenty-four hour transport; T48 = forty-eighthour transport; T7d = seven day transport; transport time = amount oftime elapsed between Time Zero when urine samples were inoculated andwhen urine samples were tested for a given parameter. ^(b)Degree ofchange measured from Time Zero: using the scales given for eachurinalysis test result listed below, 1° = a change of one step up ordown the scale, e.g., for glucose with 250 mg/dl at Time Zero, change to100 mg/dl or 500 mg/dl; 2° = a change of two steps up or down the scale,e.g., for glucose with 250 mg/dl at Time Zero, change to negative or1000+ mg/dl; 3° = a change of three steps up or down the scale, e.g., #for glucose with 100 mg/dl at Time Zero, change to 1000+ mg/dl; and 4° =a change of four steps up or down the scale, e.g., for glucose with1000+ mg/dl at Time Zero, change to negative. ^(c)Possible urinalysistest results for glucose: negative, 100, 250, 500, or 1000+ mg/dl.^(d)Possible urinalysis test results for bilirubin: negative, small,moderate, or large. ^(e)Possible urinalysis test results for ketone:negative, trace, small, moderate, or large. ^(f)Possible urinalysis testresults for specific gravity: ≧1.005, 1.010, 1.015, 1.020, 1.025, or≧1.030. ^(g)Possible urinalysis test results for pH: ≧5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, or ≧9.0. ^(h)Possible urinalysis test results forprotein: negative, trace, 30, 100, 300, or 1000+ mg/dl. ^(i)Possibleurinalysis test results for urobilinogen: 0.2, 1, 2, 4, or 8 mg/dl.^(j)Possible urinalysis test results for nitrites: negative or positive.^(k)Possible urinalysis test results for blood: negative, trace, Small,moderate or large. ^(l)Possible urinalysis test results for leukocytes:negative, trace, small, moderate or large.

Another group of liquid stabilizing transport/preservative formulationswhich are inert to plastics, rubber, and silicone, hereinafter referredto as “Chemistat III”, have been found, comprising an antimicrobialorganic acid, boric acid, and a biguanide. Preferably, Chemistat IIIcomprises an antimicrobial propionate, boric acid, and a biguanide. Mostpreferably, Chemistat III formulations comprise from about 2.5 mg/ml toabout 7.5 mg/ml sodium proprionate, from about 0.1 mg/ml to about 10mg/ml boric acid, and from about 0.01 mg/ml to about 0.1 mg/mlchlorhexidine, and any combination thereof. For preservation of urinesamples, a preferred Chemistat III formulation comprises 6.25 mg/mlsodium proprionate, 0.5 mg/ml boric acid, and 0.025 mg/ml chlorhexidine.

EXAMPLE 3 Effectiveness of Chemistat III Formulations

Chemistat III formulations were shown to preserve urine samplescontaining significant microbial contamination and at the same timemaintain the chemical and physical properties of the urine samples.

Table VIII gives the antimicrobial data for the individual components ofChemistat III. As previously discussed, chlorhexidine alone had cidalactivity against Escherichia coli and Candida albicans at 0.0075 mg/ml;Enterobacter aerogenes, at 0.01 mg/ml; Proteus vulgaris and Klebsiellapneumoniae, at 0.02 mg/ml; showed variable cidal activity againstEnterococcus faecalis at 0.05-0.8 mg/ml; and was not cidal againstPseudomonas aeruginosa at up to 0.05 mg/ml. Sodium propionate wasinhibitory against Pseudomonas aeruginosa and Enterococcus faecalis at10 mg/ml, but was ineffective against Escherichia coli, Proteusvulgaris, Candida albicans, Klebsiella pneumoniae, and Enterobacteraerogenes at up to 10 mg/ml. Boric acid was ineffective againstEscherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, andEnterobacter aerogenes. Thus, the components of the Chemistat IIIformulations when used alone were unable to control the growth of themicroorganisms of interest.

TABLE VIII Antimicrobial Activity for Individual Chemistat IIIComponents Survival Values^(a) after 24 hours at Room Temperature forRepresentative Organisms^(b) Component esco psae prvu enfa caal klpnenae Chlorhexidine (0.005 mg/ml) 0.003 tntc 1.115 5.308 tntcChlorhexidine (0.0075 mg/ml) 0.000 4.049 0.598 0.000 6.098 Chlorhexidine(0.01 mg/ml) 0.000 0.277 0.520 0.000 Chlorhexidine (0.02 mg/ml) 0.0000.275 0.000 0.179 Chlorhexidine (0.02 mg/ml) 0.000 0.080 0.000 0.1410.000 0.000 Chlorhexidine (0.05 mg/ml) 0.000 0.046 0.000 0.000 0.000Chlorhexidine (0.1 mg/ml) 0.000 0.561 0.000 Chlorhexidine (0.8mg/ml)^(c) 0.000 0.000 0.000 Boric Acid (5 mg/ml) 0.770 1.157 2.6471.061 Sodium propionate (5 mg/ml) tntc tntc tntc tntc tntc tntc Sodiumpropionate (10 mg/ml) tntc 0.341 2.702 ^(a)Numbers indicate survivalvalues (number of organisms recovered after 24 hours incubation at roomtemperature divided by the initial control count; tntc = organisms at 24hours too numerous to count. ^(b)Organism Key: esco = Escherichia coli;psae = Pseudomonas aeruginosa; prvu = Proteus vulgaris; enfa =Enterococcus faecalis; caal = Candida albicans; klpn = Klebsiellapneumoniae; enae = Enterobacter aerogenes. ^(c)Produced a false positiveprotein upon urinalysis.

TABLE IX Antimicrobial Activity for Chemistat III Component CombinationsSurvival Values^(a) after 24 hours at Room Temperature forRepresentative Organisms^(b) Component Combinations esco psae prvu enfacaal klpn enae Chlorhexidine (0.02 mg/ml); 0.000 0.005 0.000 0.448sodium propionate (10 mg/ml) Sodium propionate (5 mg/ml); 0.000 0.3260.000 0.605 0.000 0.000 chlorhexidine (0.02 mg/ml) Boric Acid (1 mg/ml);0.000 2.304 1.231 0.000 1.861 chlorhexidine (0.005 mg/ml) Chlorhexidine(0.02 mg/ml); 0.000 0.000 0.000 0.020 sodium propionate (10 mg/ml);boric acid (5 mg/ml) ^(a)Numbers indicate survival values (number oforganisms recovered after 24 hours incubation at room temperaturedivided by the initial control count; tntc = organisms at 24 hours toonumerous to count. ^(b)Organism Key: esco = Escherichia coli; psae =Pseudomonas aeruginosa; prvu = Proteus vulgaris; enfa = Enterococcusfaecalis; caal = Candida albicans; klpn = Klebsiella pneumoniae; enae =Enterobacter aerogenes.

Table IX presents antimicrobial data for Chemistat III componentcombinations. Boric acid (1 mg/ml) and chlorhexidine (0.005 mg/ml) incombination was not cidal against Pseudomonas aeruginosa, Enterococcusfaecalis, and Enterobacter aerogenes. Likewise, sodium propionate (5mg/ml) and chlorhexidine (0.02 mg/ml) in combination was not cidalagainst Pseudomonas aeruginosa and Enterococcus faecalis. Whenchlorhexidine (0.02 mg/ml), sodium propionate (10 mg/ml) and boric acid(5 mg/ml) were combined, the composition was cidal against themicroorganisms of interest given in Table IX and did not interfere withurinalysis (data not shown).

It is to be understood that the examples presented herein are merelyrepresentative of the transport/preservative formulations of the presentinvention characterized as being cidal against the microorganisms ofinterest and not altering the chemical and physical properties of thesample, and that the scope of the invention extends to othercombinations of antimicrobial aromatic alcohols, alkyl guaiacols,biguanides, organic acids, lipid membrane-disrupting agents, and boricacid derivatives. While the examples demonstrated the stabilization ofurine samples contaminated with microorganisms commonly found in urine,it is to be further understood that the transport/preservative system ofthe present invention can be applied to the preservation of otherpatient samples, bodily fluids, biological reagents, therapeutics, andpersonal care products, and that preferred combinations andconcentrations of components in the formulations of the presentinvention for these applications can be easily determined by followingthe teachings presented in the examples.

The transport/preservative system of the present invention maintains thechemical and physical integrity of patient specimens or bodily fluidsduring extended transport. Examples of the patient specimens for whichthe transport system is effective include but are not limited to urine,saliva, sputum, and tissue biopsies.

Samples submitted for DNA or RNA analysis or PCR probe technology canalso be preserved without the transport/preservative system of thepresent invention. While major efforts have been made in DNA/RNAanalysis and PCR probe technology to block inhibitors of the criticalreactions and to block the negative action of DNase and RNase. However,no efforts have been made to block the potential negative effects ofgrowth and/or metabolism of organisms in the sample. Growth ormetabolism can drastically affect both probe technology and PCR, becauseit can alter the chemical properties of the sample. Also, live organismscan excrete DNase and RNase. These samples must be processed immediatelyafter sampling, or they must have a suitable preservative added whichwill kill the microorganisms. The cidal action of thetransport/preservative system of the present invention provides thenecessary killing action.

The transport/preservative system of the present invention can also beused for stabilizing biological reagents. For example, numerous assaymethodologies routinely used for both diagnostic and research purposesrequire reagents that contain substances capable of supporting thegrowth of contaminating microorganisms. Thus, the reagents aresusceptible to both physical and chemical degradation caused by thegrowth and/or metabolism of contaminating microorganisms. Examples ofsuch reagents include but are not limited to starting materials,catalysts, cofactors, surface-active agents, nuclei acid, protein,carbohydrate, and lipid standards, antigen-antibody reagents, geneticmarkers and probes including those used in PCR technology, and detectioncompositions. The chemical composition of such reagents include but arenot limited to amino acids, carbohydrates, fatty acids, nucleic acids,proteins, polysaccharides, lipids, sugars, and mixtures and/or complexthereof.

The present invention also includes a device which comprises anaccessible, sealable enclosure for containing and preserving a samplewhich may contain microorganisms which enclosure contains a compositionfree of toxins including mercury, mercury containing compounds,formaldehyde, formaldehyde releasing compounds, and azides which areunsuitable for use in high volume processing and comprises a biguanideand one or more additional antimicrobial agents, wherein the compositionis cidal to microorganisms when present in the sample. This device isfor the preservation of a sample selected from a patient sample, abodily fluid such as urine, tissue specimen, and reagent, a biologicalreagent, a therapeutic, or a personal care product. The preferredbiguanide in the device is chlorhexidine. One preferred compositionuseful in the device comprises an aromatic alcohol such as 2-phenylethanol, a terpenoid such as isoeugenol, and a biguanide such aschlorhexidine. Another preferred composition comprises an organic acidsuch as sodium propionate, a parahydroxybenzoate such as ethylparahydroxybenzoate, and a biguanide such as chlorhexidine. Yet anotherpreferred composition comprises a propionate such as sodium propionate,boric acid and/or boric acid derivative, and a biguanide such aschlorhexidine.

The transport/preservative system of the present invention is effectivein the preservation of aqueous or alcoholic therapeutics includingpharmaceuticals, nutraceuticals and over-the-counter preparations whichcontain substances capable of supporting the growth of contaminatingmicroorganisms. Examples of therapeutics preserved by the presentinvention include but are not limited to vaccines, antigen preparationsfor allergy treatment, hormone preparations, insulin, nasal sprays,liquid cough and cold remedies, liquid allergy medications, and liquidvitamin and nutrient preparations.

Personal care products can also be preserved from chemical and/orphysical degradation using the transport/preservation system of thepresent invention. Examples of personal care products preserved by thepresent invention include but are not limited to cosmetics, handcleansers, lotions, shampoos and contact lens solutions.

The transport/preservation formulations can also be used as sanitizersfor surfaces, equipment, and appliances. Upon application of a sanitizercomprising the transport/preservative formulation, a residue remainswhich reduces the microbial bioburden over time.

1. A system for preserving a sample which may contain microorganisms,said system including a patient biological specimen and a composition,including said specimen, having (a) a concentration of about 0.01 toabout 0.1 mg/ml of a biguanide and (b) at least one other antimicrobialagent, said composition being cidal to said microorganisms when presentin said patient biological specimen.
 2. The system of claim 1, whereinsaid biguanide is chlorhexidine.
 3. The system of claim 2, wherein saidmicroorganisms have cell membranes and said cell membranes have aselective permeability; and said at least one other antimicrobial agentcomprises a compound that reduces said selective permeability.
 4. Thesystem of claim 3, wherein said at least one other antimicrobial agentcomprises an aromatic alcohol.
 5. The system of claim 4, wherein saidaromatic alcohol is 2-phenyl ethanol.
 6. The system of claim 5, whereinsaid composition further comprises a terpenoid.
 7. The system of claim6, wherein said terpenoid is isoeugenol.
 8. The system of claim 4,wherein said composition further comprises a terpenoid.
 9. The system ofclaim 8, wherein said terpenoid is isoeugenol.
 10. The system of claim2, wherein said at least one other antimicrobial agent comprises apropionate.
 11. The system of claim 10, wherein said propionate issodium propionate.
 12. The system of claim 11, wherein said compositionfurther comprises a parahydroxybenzoate.
 13. The system of claim 12,wherein said parahydroxybenzoate is ethyl parahydroxybenzoate.
 14. Thesystem of claim 11, wherein said composition further comprises boricacid or a boric acid derivative.
 15. The system of claim 10, whereinsaid composition further comprises a parahydroxybenzoate.
 16. The systemof claim 15, wherein said parahydroxybenzoate is ethylparahydroxybenzoate.
 17. The system of claim 10, wherein saidcomposition further comprises boric acid or a boric acid derivative. 18.The system of claim 2, wherein said at least one other antimicrobialagent comprises boric acid or a boric acid derivative.
 19. The system ofclaim 1, wherein said microorganisms have cell membranes and said cellmembranes have a selective permeability; and said at least one otherantimicrobial agent comprises a compound that reduces said selectivepermeability.
 20. The system of claim 19, wherein said at least oneother antimicrobial agent comprises an aromatic alcohol.
 21. The systemof claim 20, wherein said aromatic alcohol is 2-phenyl ethanol.
 22. Thesystem of claim 21, wherein said composition further comprises aterpenoid.
 23. The system of claim 22, wherein said terpenoid isisoeugenol.
 24. The system of claim 20, wherein said composition furthercomprises a terpenoid.
 25. The system of claim 24, wherein saidterpenoid is isoeugenol.
 26. The system of claim 1, wherein said atleast one other antimicrobial agent comprises a propionate.
 27. Thesystem of claim 26, wherein said propionate is sodium propionate. 28.The system of claim 27, wherein said composition further comprises aparahydroxybenzoate.
 29. The system of claim 28, wherein saidparahydroxybenzoate is ethyl parahydroxybenzoate.
 30. The system ofclaim 27, wherein said composition further comprises boric acid or aboric acid derivative.
 31. The system of claim 26, wherein saidcomposition further comprises a parahydroxybenzoate.
 32. The system ofclaim 31, wherein said parahydroxybenzoate is ethyl parahydroxybenzoate.33. The system of claim 26, wherein said composition further comprisesboric acid or a boric acid derivative.
 34. The system of claim 1,wherein said at least one other antimicrobial agent comprises boric acidor a boric acid derivative.
 35. The system of claims 1, 2, 26, 10, 27,11, 31, 15, 28, 12, 32, 16, 29, 13, 34, 18, 33, 17, 30 or 14, whereinsaid patient biological specimen is selected from the group consistingof: (a) bodily fluids, including urine, saliva and sputum; and (b)biological tissue specimens.
 36. A system for preserving a urine samplewhich may contain microorganisms, said system comprising: a compositionincluding said sample having a concentration of (a) about 0.01 to 0.1mg/ml of chlorhexidine and (b) about 0.1 to 0.7 mg/ml ethyl paraben. 37.A system for preserving a urine sample which may contain microorganisms,said system comprising: a composition including said sample having aconcentration of (a) about 0.01 to 0.1 mg/ml chlorhexidine and (b) about1 to 10 mg/ml sodium propionate.
 38. The system of claim 37, whereinsaid composition including said sample has a concentration of about 0.1to 1.0 mg/ml ethyl paraben.
 39. The system of claim 38 wherein saidcomposition including said sample has a concentration of about 2.5 mg/mlto about 7.5 mg/ml sodium propionate, about 0.1 mg/ml to about 1 mg/mlethyl parahydroxybenzoate, and about 0.01 mg/ml to about 0.1 mg/mlchlorhexidine.
 40. The system of claim 37, wherein said compositionincluding said sample has a concentration of about 0.25 to 1 mg/ml boricacid.
 41. A system for preserving a urine sample which may containmicroorganisms, said system comprising: a composition including saidsample having a concentration of (a) about 0.01 to 0.1 mg/ml ofchlorhexidine, (b) about 0 to 2.5 μl/ml 2 phenyl ethanol, and (c) about0.2 to 1.5 μl/ml isoeugenol.
 42. A system for preserving a urine samplewhich may contain microorganisms, said system comprising: a compositionconsisting essentially of chlorhexidine and ethyl paraben, saidcomposition including said sample having a concentration of (a) about0.01 to 0.1 mg/ml of chlorhexidine and (b) about 0.1 to 1 mg/ml ethylparaben.
 43. A system for preserving a urine sample which may containmicroorganisms, said system comprising: a composition consistingessentially of chlorhexidine and sodium propionate, said compositionincluding said sample having a concentration of (a) about 0.01 to 0.1mg/ml chlorhexidine and (b) about 1 to 10 mg/ml sodium propionate.
 44. Asystem for preserving a urine sample which may contain microorganisms,said system comprising: a composition consisting essentially ofchlorhexidine, sodium propionate and ethyl paraben, said compositionincluding said sample having a concentration of (a) about 0.01 to 0.1mg/ml chlorhexidine, (b) about 1 to 10 mg/ml sodium propionate and (c)about 0.1 to 1 mg/ml ethyl paraben.
 45. A system for preserving a urinesample which may contain microorganisms, said system comprising: acomposition consisting essentially of chlorhexidine, sodium propionateand boric acid, said composition including said sample having aconcentration of (a) about 0.01 to 0.1 mg/ml chlorhexidine, (b) about 1to 10 mg/ml sodium propionate and (c) about 0.25 to 1 mg/ml boric acid.46. A system for preserving a urine sample which may containmicroorganisms, said system comprising: a composition consistingessentially of chlorhexidine, 2 phenyl ethanol, and isoeugenol, saidcomposition including said sample having a concentration of (a) about0.01 to 0.1 mg/ml of chlorhexidine, (b) about 0 to 2.5 μl/ml 2 phenylethanol, and (c) about 0.2 to 1.5 μl/ml isoeugenol.
 47. A system forpreserving a urine sample which may contain microorganisms, said systemcomprising: a composition consisting essentially of chlorhexidine,sodium propionate, and boric acid, said composition including saidsample having a concentration of (a) about 0.01 to 0.1 mg/ml ofchlorhexidine, (b) about 2.5 to 7.5 mg/ml sodium propionate, and (c)about 0.1 mg/ml to about 10 mg/ml boric acid.